Since the neuronal .alpha.Bgt binding proteins of the present invention and nicotinic acetylcholine receptors and certain other receptor types appear to be members of the same gene superfamily, it seems appropriate to start the review of background art with a brief overview of our recent knowledge of the known types of such receptors.
The study of cell to cell contacts known as synapses, has long been a focal point of neuroscience research. This is particularly true of the neuromuscular synapse (often called neuromuscular junction), which occurs at the point of nerve to muscle contact, primarily because of its accessibility to biochemical and electrophysiological techniques and because of its elegant, well defined structure. Much of this research has concentrated on acetylcholine receptors because they are a critical link in transmission of signals from nerves to muscles. Action potentials propagated along a motor nerve axon from the spinal cord depolarize the nerve ending causing it to release a chemical neurotransmitter, acetylcholine. Acetylcholine binds to nicotinic acetylcholine receptor proteins located on post-synaptic muscle cells, triggering the brief opening of a cation channel through the receptor molecule and across the postsynaptic membrane. The resulting flux of cations across the membrane triggers an action potential that is propagated along the surface membrane of the muscle, thereby completing neuromuscular transmission and ultimately causing contraction of the muscle.
Several lines of evidence demonstrate that nicotinic acetylcholine receptors (AChRs) of muscle, brain and ganglia, belong to the same family and function as acetylcholine (ACh)-gated cation channels.
The most well-studied member of this family is the muscle-type AChR of the electric organs of Torpedo californica and related species, because electric organs contain much larger amounts of nicotinic acetylcholine receptors than muscle. Muscle-type nicotinic AChRs are composed of four kinds of subunits, including two .alpha. subunits which contain the ACh-binding sites, and must be both liganded to efficiently trigger opening of the cation channel, and one each of .beta., .gamma. and .delta. subunits. The subunits are oriented like barrel staves in the order of .alpha..beta..alpha..gamma..delta. around the central cation channel whose opening is regulated by binding of ACh to the .alpha. subunits. All of the AChR subunits have sequence homologies throughout their lengths which suggests that they evolved by gene duplication from a common ancestor and have similar basic structures. Evolution of muscle-type AChRs has been quite conservative; there is 80% sequence homology between .alpha. subunits of AChRs of the electric organs of Torpedo californica and human muscle, and about 55% homology of the other subunits between these species. However, AChRs of different origin are much more antigenically distinct than their sequence homologies would suggest. A concise review of the structure and properties of muscle-type nicotinic AChRs is, for example, provided by Lindstrom et al, Mol. Neurobiol. 1(4), 281 (1987). Study of these receptors has been greatly enhanced by the availability of suitable molecular probes, in particular .alpha.-Bungarotoxin (.alpha.Bgt). .alpha.Bgt binds with great affinity to the ACh-binding sites of muscle-type nicotinic AChRs thereby inhibiting their function, and can be used as a histological label, affinity column ligand, and probe for the ACh-binding site of the purified AChRs. .alpha.Bgt satisfies three criteria that certify it as a ligand for muscle-type AChRs: (i) its binding to muscle is saturable and agonists and antagonists of ACh compete for this binding; (ii) it binds to cells and regions of cells that respond physiologically to ACh; (iii) it blocks the physiological response of muscle to ACh.
Nicotinic acetylcholine receptors on neurons (neuronal nicotinic AChRs), that are members of the same ligand-gated cation channel family as muscle-type nicotinic AChRs, have originally been much less well characterized than have their muscle-type relatives. This was partly because there were no neural systems as convenient as muscle and there was no neural system that provided as much AChR as electric organs. Also, studies were hampered by lack of suitable molecular probes. As mentioned before, the snake toxin .alpha.Bgt has proven an invaluable tool for characterizing muscle-type AChRs. Although it was originally inferred that .alpha.Bgt binds to neuronal AChRs, later works made it clear that the .alpha.Bgt receptors and neuronal acetylcholine receptors are not equivalent [see e.g. Carbonetto et al., Proc. Natl. Acad Sci USA 75, 2 (1978) on the non-equivalence of .alpha.-Bungarotoxin receptors and acetylcholine receptors in chick sympathetic neurons]. Since .alpha.Bgt does not bind to neuronal nicotinic AChRs, it is not a useful probe for structural studies. More recently, monoclonal antibodies (mAbs) have proven extremely useful in studying neuronal AChRs. Immunoaffinity-purified neuronal AChRs were found to consist of only two kinds of subunits. The larger subunit was identified as the ACh-binding subunit by nicotine-blockable 4-(N-maleimido)benzyltri[.sup.3 H]-methylammonium iodide (MBTA) labeling after reduction, suggesting that it contains cysteines homologous to cysteines 192,193 of the muscle-type Torpedo AChR .alpha. subunits. The lower molecular weight subunit is often called the .beta. subunit, but, more informatively, is also referred to as the structural subunit. The subunit stoichiometry is uncertain, but there are indications that it may be x.sub.2 y.sub.2 (x and y standing for the ACh-binding and structural subunits, respectively). These subunits exhibit sequence homologies which indicate that they belong to the same gene family as the subunits of muscle-type nicotinic AChRs. There appear to be several subtypes of these AChRs, including at least four kinds of ACh-binding subunits and two kinds of structural subunits. Some different subtypes may use the same structural subunit and differ in which ACh-binding subunit they use. Schoepfer et al., Molecular Biology of Neuroreceptors and Ion Channels, NATO-ASI Series, H vol. 32, pp. 37-53 (1989), A. Maelicke (Ed.), Springer-Verlag, Heidelberg, and the references cited therein provide a comparison of the structures of muscle and neuronal nicotinic AChRs. Further details of the character of neuronal nicotinic AChRs from different sources (chicken brain, rat, bovine, human, etc.) and of the distribution of the two types of subunits can be found, for example, in Whiting and Lindstrom, J. Neurosci. 8(9), 3395 (1988), and Wada et al., J. Comp. Neurol. 284, 314 (1989).
Some receptors for glycine and .gamma.-aminobutyric acid, such as the strychnine-binding glycine receptor of the spinal cord and the brain .gamma.-aminobutyric acid.sub.A (GABA.sub.A), are ligand-gated anion channels which appear to be more distant members of the same receptor superfamily which includes nicotinic receptors and may include other ligand-gated ion channels such as some receptors for glutamate and serotonin. Barnard et al., Trends Neurosci. 10, 502 (1987) compare the primary structures of the brain GABA.sub.A receptor, one of the subunits of the spinal cord glycine receptor and those of muscle-type and neuronal nicotinic AChRs.
Other neurotransmitter receptors whose subunit cDNAs have been cloned, are characterized as receptors without intrinsic ion channels. These receptors act through a GTP-binding protein and various enzymes to produce second messengers. All these receptors have a single subunit with sequence homology to rhodopsin. Typical representatives of this group are muscarinic AChRs and adrenergic receptors. Muscarinic AChRs are distinguished by their differential sensitivity to the alkaloid compound muscarine, and regulate a broad range of physiological and biochemical activities throughout the central and autonomic nervous systems via the activation of guanine nucleotide binding (G) proteins. A good summary of the recent knowledge about the structural and biochemical diversity of muscarinic AChRs is provided by Peralta et al., TIPS-February supplement, 6 (1988). Subtype-specific agonist and antagonist binding characteristics of chimeric .beta..sub.1 and .beta..sub.2 -adrenergic receptors are disclosed by Frielle et al., Proc. Natl. Acad. Sci. USA 85, 9494 (1988).
As mentioned before, the neuronal AChRs exhibit high affinity for nicotine and other cholinergic agonists, but do not bind .beta.Bgt. Binding studies using the technique of autoradiography to produce detailed maps of [.sup.3 H]nicotine, [.sup.3 H]ACh and [.sup.125 I].alpha.Bgt labeling in near-adjacent sections of rat brain have revealed that whereas [.sup.3 H]nicotine and [.sup.3 H]ACh bind with strikingly similar pattern, there is remarkably little overlap with [.sup.125 I].alpha.Bgt labeling [Clarke et al., Journal Neurosci. 5, 1307 (1985)]. Based upon their experiments, that were in excellent agreement with previous works on .alpha.Bgt binding in rodent brain, Clarke et al. concluded that .alpha.Bgt may label a new kind of nicotinic receptors, that is clearly different from nicotinic AChRs. These receptors are usually referred to in the literature as neuronal .alpha.Bgt binding proteins (.alpha.BgtBPs). Their concentration throughout the vertebrate brain is considerably higher than that of neuronal AChRs. A number of laboratories have documented the existence of .alpha.BgtBPs in sympathetic ganglion membranes and membrane fragments derived from vertebrate brain. Since the ability of cholinergic ligands to effect receptor function remained unknown and .alpha.Bgt was found to have no effect on agonist-induced activation of acetylcholine receptors in these tissues, the identity of the toxin-binding component was entirely uncertain. The binding of .alpha.Bgt to a clonal rat sympathetic nerve cell line was described by Patrick, J. and Stallcup, W., J. Biol Chem. 252, 8629 (1977). The binding was found to be saturable and was inhibited by a variety a cholinergic agonists and antagonists. In assays determining the binding constants for various cholinergic ligands no correlation was found between their ability to affect cholinergic function and to inhibit binding of .alpha.Bgt. It was found that the site at which cholinergic ligands affect AChR function is different from the site at which cholinergic ligands inhibit .alpha.Bgt binding. The authors concluded that the .alpha.Bgt binding component is probably a single molecular species of an integral membrane protein which is different from the functional neuronal nicotinic AChR.
Carbonetto et al., Supra delivered evidence that the .alpha.Bgt receptors in chick sympathetic neurons are not neuronal AChRs. Similar results were reported by Smith et al., J. Neurosci. 3, 2395 (1983) and Jacob et al., J. Neurosci. 3, 260 (1983) on chick ciliary ganglion neurons. The major findings in these articles are that neuronal levels of ACh sensitivity do not correlate with .alpha.Bgt binding sites, and in the case of chick ciliary ganglion cells the .alpha.BgtBPs, unlike neuronal nicotinic acetylcholine receptors, are not located at synapses. Although Smith et al. have ruled out some trivial reasons for the lack of correlation between ACh sensitivity and .alpha.Bgt binding, they or Jacob et al., Supra provide no explanation of the function of the .alpha.Bgt binding site.
Conti-Tronconi et al., Proc. Natl. Acad. Sci. USA 82 (1985) purified .alpha.Bgt-binding proteins from chick optic lobe and brain under conditions that were designed to minimize proteolysis. Five different peptides with molecular weights ranging between about 48,000 and about 72,000 were separated by gel electrophoresis and submitted to amino-terminal amino acid sequencing. The amino-terminal amino acid sequence of the 48,000 molecular weight subunit was found to be highly homologous to the sequences of known .alpha. subunits of peripheral AChRs from Torpedo electroplax, Electrophorus electroplax and muscle, and calf muscle. Amino-terminal amino acid sequence analysis of the other isolated protein fragments did not yield any signal above the high background consistently present, indicating that these fragments probably had blocked amino termini. Although there are some indications that the protein fragments not sequenced may be part of the same receptor, the subunit structure of brain .alpha.BgtBPs has not been established. Conti-Tronconi et al., Supra add to the confusion concerning the terminology, identity, and function of vertebrate .alpha.Bgt binding proteins by concluding that brain .alpha.BgtBPs are nicotinic AChRs.
Whiting, P. and Lindstrom, J., Proc. Natl. Acad. Sci. USA 84, 595 (1987) report the purification of an .alpha.BgtBP from rat brain and the identification of four kinds of subunits. The four polypeptides separated by affinity-purification of the .alpha.BgtBP were similar in their apparent molecular weights to the .alpha., .beta., .gamma. and .delta. subunits of muscle-type nicotinic AChRs. In the absence of antibody probes for the toxin binding protein, the authors could not unequivocally demonstrate that each of the four polypeptides were true constituents of the same macromolecule.
Like muscle and neuronal AChRs, .alpha.BgtBPs can be affinity labeled with MBTA after reduction with dithiothreitol (DTT). In chicken brain this labels a subunit of apparent molecular weight of 45,000. This suggests that, as in nicotinic AChRs, there is a readily reducible disulfide bond near the ACh binding site. The ACh-binding subunits of all nicotinic AChRs characterized to date exhibit a pair of cysteines homologous to the disulfide-bound pair at .alpha.192,193 in muscle-type AChR .alpha. subunits which are known to be affinity labeled by MBTA.
Norman et al., Proc. Natl. Acad. Sci. USA 79, 1321 (1982) report the isolation of the .alpha.BgtBP from chick optic lobe as a pure glycoprotein and compare this protein with the .alpha.Bgt-binding component isolated from the rest of the chick brain. The authors conclude that the .alpha.BgtBP from chick optic lobe and the .alpha.BgtBP from the rest of the brain appear similar or identical by a series of criteria and are both related to peripheral AChRs. For further information see, for example: Kao et al., J. Biol. Chem. 259, 11662 (1984); Kao et al., J. Biol. Chem. 261, 8085 (1986); Whiting, P. and Lindstrom, J., FEBS Lett. 213(1), 55 (1987); and Whiting et al., FEBS Lett. 219(2), 459 (1987).
The selected literature references cited hereinabove illustrate the controversial nature of our present knowledge about .alpha.BgtBPs. The following main observations have been made so far:
Vertebrate (avian and mammalian) brains contain both nicotinic AChRs which have high affinity for nicotine and acetylcholine but not for .alpha.Bgt, and distinct .alpha.Bgt-binding sites. There are more .alpha.BgtBPs than neuronal AChRs.
In some cases (chick ciliary ganglion cells, chick sympathetic ganglion cells, rat PC12 pheochromocytoma cells), .alpha.BgtBPs appear to be made by cells which also produce neuronal nicotinic AChRs, but these .alpha.BgtBPs do not appear to function as ACh-gated cation channels.
In the case of chick ciliary ganglion cells, the .alpha.BgtBPs are not located at synapses.
There are some claims of vertebrate neuronal AChRs whose function is blocked by .alpha.Bgt, but so far they are not supported by conclusive evidence.
The .alpha.Bgt-binding proteins from brains of chicks, and rats (similarly to muscle-type AChRs, and neuronal AChRs) can be affinity labeled with BAC and MBTA, after reduction with DTT. This suggests that amino acid residues homologous to Cys .alpha.192-193 of electric organ and muscle AChRs may have been conserved in the members of what appears to be an extended gene family consisting of muscle-type AChRs, neuronal AChR subtypes, and neuronal .alpha.BgtBPs.
The partial N-terminal amino acid sequence of one .alpha.BgtBP subunit of apparent molecular weight of 48,000 (Conti-Tronconi, et al., Supra) from chicken brain exhibits sequence homology with AChR subunits. This, along with pharmacological properties, is another indication that muscle-type and neuronal nicotinic AChRs and .alpha.BgtBPs are probably members of the same gene superfamily.
It is certain that neuronal .alpha.BgtBPs are functionally, structurally, immunologically, histologically, and pharmacologically distinct from functional vertebrate AChRs which do not bind .alpha.Bgt.
However, the function of the .alpha.Bgt-binding sites remains obscure. There is a large body of evidence indicating that the .alpha.BgtBPs which have been most carefully studied are not ACh-gated cation channels. Although there are indications that they may have several subunits, the subunit structure of .alpha.BgtBPs is unclear. The only information available in the art concerning the sequence of .alpha.BgtBP subunits, is a short N-terminal amino acid sequence of a polypeptide of the .alpha.BgtBP from chicken brain reported by Conti-Tronconi, et al., Supra.